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Respiratory syncytial virus (RSV) is a common childhood infection that in young infants can progress into severe bronchiolitis and pneumonia. Disease pathogenesis results from both viral mediated and host immune processes of which alveolar macrophages play an important part. Here,we investigated the role of different types of alveolar macrophages on RSV infection using an in vitro co-culture model involving primary tissue-derived human bronchial epithelial cells (HBECs) and human blood monocyte-derived M0-like,M1-like,or M2-like macrophages. It was hypothesized that the in vitro model would recapitulate previous in vivo findings of a protective effect of macrophages against RSV infection. It was found that macrophages maintained their phenotype for the 72-hour co-culture time period and the bronchial epithelial cells were unaffected by the macrophage media. HBEC infection with RSV was decreased by M1-like macrophages but enhanced by M0- or M2-like macrophages. The medium used during the co-culture also impacted the outcome of the infection. This work demonstrates that alveolar macrophage phenotypes may have differential roles during epithelial RSV infection,and demonstrates that an in vitro co-culture model could be used to further investigate the roles of macrophages during bronchial viral infection. View Publication

The role of negative checkpoint regulators (NCRs) in human health and disease cannot be overstated. V-domain Ig-containing Suppressor of T-cell Activation (VISTA) is an Ig superfamily protein predominantly expressed within the hematopoietic compartment and has been studied for its role in the negative regulation of T cell responses. The findings presented in this study show that,unlike all other NCRs,VISTA deficiency dramatically impacts on macrophage cytokine and chemokine production,as well as the chemotactic response of VISTA-deficient macrophages. A select group of inflammatory chemokines,including CCL2,CCL3,CCL4,and CCL5,was strikingly elevated in culture supernatants from VISTA KO macrophages. VISTA deficiency also altered chemokine receptor recycling and profoundly disrupted myeloid chemotaxis. The impact of VISTA deficiency on chemotaxis in vivo was apparent with the reduced ability of both KO macrophages and MDSCs to migrate to the tumor microenvironment. This is the first demonstration of an NCR impacting on myeloid mediator production and chemotaxis,and will guide the use of anti-VISTA therapeutics to manipulate the chemotaxis of inflammatory macrophages or immunosuppressive MDSCs in inflammatory diseases and cancer. View Publication

Acute megakaryoblastic leukemia (AMKL) represents ∼10{\%} of pediatric acute myeloid leukemia cases and typically affects young children ({\textless}3 years of age). It remains plagued with extremely poor treatment outcomes ({\textless}40{\%} cure rates),mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60{\%} to 70{\%} of cases and include nucleoporin 98 (NUP98) gene rearrangements,most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking,and patient samples are rare,further limiting biomarkers and drug discovery. To overcome these impediments,we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest,sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia,including AMKL,that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP,MPIG6B,and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib,a clinically approved JAK2 inhibitor. Overall,synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia,which lack effective and rationally designed treatments. View Publication

A rapidly increasing number of reports on dysregulated long intergenic non-coding RNA (lincRNA) expression across numerous types of cancers indicates that aberrant lincRNA expression may be a major contributor to tumorigenesis. Marek's disease (MD) is a T cell lymphoma of chickens induced by Marek's disease virus (MDV). Although we have investigated the roles of lincRNAs in bursa tissue of MDV-infected chickens in previous studies,the molecular mechanisms of lincRNA functions in T cells remain poorly understood. In the present study,Linc-GALMD1 was identified from CD4+ T cells and MSB1 cells,and its expression was significantly downregulated in MD-resistant line of birds in response to MDV challenge. Furthermore,loss-of-function experiments indicated that linc-GALMD1 significantly affected the expression of 290 genes in trans. Through integrated analysis of differentially expressed genes (DEGs) induced by MDV and linc-GALMD1,we found that IGLL1 gene expression levels had a positive correlation with the degree of MD infection and could potentially serve as an indicator for clinical diagnosis of MD. Moreover,an interaction between MDV and linc-GALMD1 was also observed. Accordingly,chicken embryonic fibroblast cells were inoculated with MDV with and without the linc-GALMD1 knockdown,and the data showed that linc-GALMD1 could repress MDV gene expression during the course of MDV infection. These findings uncovered a role of linc-GALMD1 as a viral gene regulator and suggested a function of linc-GALMD1 contributing to tumor suppression by coordinating expression of MDV genes and tumor-related genes and regulating immune responses to MDV infection. View Publication

Early studies in exercise immunology suggested acute bouts of exercise had an immunosuppressive effect in human subjects. However,recent data,show acute bouts of combined aerobic and resistance training increase both lymphocyte activation and proliferation. We quantified resistance exercise-induced changes in the activation state of CD4+ T lymphocytes via surface protein expression and using a medically relevant model of infection (HIV-1). Using a randomized cross-over design,10 untrained subjects completed a control and exercise session. The control session consisted of 30-min seated rest while the exercise session entailed 3 sets × 10 repetitions of back squat,leg press,and leg extensions at 70{\%} 1-RM with 2-min rest between each set. Venous blood samples were obtained pre/post each session. CD4+ T lymphocytes were isolated from whole blood by negative selection. Expression of activation markers (CD69 {\&} CD25) in both nonstimulated and stimulated (costimulation through CD3+ CD28) cells were assessed by flow cytometry. Resistance exercised-induced effects on intracellular activation was further evaluated via in vitro infection with HIV-1. Nonstimulated CD4+ T lymphocytes obtained postexercise exhibited elevated CD25 expression following 24 h in culture. Enhanced HIV-1 replication was observed in cells obtained postexercise. Our results demonstrate that an acute bout of resistance exercise increases the activation state of CD4+ T lymphocytes and results in a greater susceptibility to HIV-1 infection in vitro. These findings offer further evidence that exercise induces activation of T lymphocytes and provides a foundation for the use of medically relevant pathogens as indirect measures of intracellular activation. View Publication

Secreted factors derived from Lactobacillus are able to dampen pro-inflammatory cytokine responses. Still,the nature of these components and the underlying mechanisms remain elusive. Here,we aimed to identify the components and the mechanism involved in the Lactobacillus-mediated modulation of immune cell activation. PBMC were stimulated in the presence of the cell free supernatants (CFS) of cultured Lactobacillus rhamnosus GG and Lactobacillus reuteri DSM 17938,followed by evaluation of cytokine responses. We show that lactobacilli-CFS effectively dampen induced IFN-$\gamma$ and IL-17A responses from T- and NK cells in a monocyte dependent manner by a soluble factor. A proteomic array analysis highlighted Lactobacillus-induced IL-1 receptor antagonist (ra) as a potential candidate responsible for the IFN-$\gamma$ dampening activity. Indeed,addition of recombinant IL-1ra to stimulated PBMC resulted in reduced IFN-$\gamma$ production. Further characterization of the lactobacilli-CFS revealed the presence of extracellular membrane vesicles with a similar immune regulatory activity to that observed with the lactobacilli-CFS. In conclusion,we have shown that lactobacilli produce extracellular MVs,which are able to dampen pro-inflammatory cytokine responses in a monocyte-dependent manner. View Publication

Tissue-resident memory CD8+ T (Trm) cells mediate potent local innate and adaptive immune responses and play a central role against solid tumors. However,whether Trm cells cross-talk with dendritic cells (DCs) to support anti-tumor immunity remains unclear. Here we show that antigen-specific activation of skin Trm cells leads to maturation and migration to draining lymph nodes of cross-presenting dermal DCs. Tumor rejection mediated by Trm cells triggers the spread of cytotoxic CD8+ T cell responses against tumor-derived neo- and self-antigens via dermal DCs. These responses suppress the growth of intradermal tumors and disseminated melanoma lacking the Trm cell-targeted epitope. Moreover,analysis of RNA sequencing data from human melanoma tumors reveals that enrichment of a Trm cell gene signature associates with DC activation and improved survival. This work unveils the ability of Trm cells to amplify the breath of cytotoxic CD8+ T cell responses through DCs,thereby strengthening anti-tumor immunity. View Publication

Despite implementation of virtual crossmatches,flow cytometry crossmatches (FCXM) are still used by many transplant centers to determine immunological risk before kidney transplantation. To determine if common profiles of patients prone to false positive FCXM exist,we examined the demographics and native diseases of kidney patients tested with autologous FCXM (n = 480). Improvements to FCXM and cell isolation methods significantly reduced the positive rate from 15.1{\%} to 5.3{\%}. Patients with native diseases considered 'immunological' (vasculitis,lupus,IgA nephropathy) had more positive autologous FCXM (OR = 3.36,p = 0.003) vs. patients with all other diseases. Patients who were tested using our updated method (n = 321) still showed that these immunological diseases were a significant predictor for positive autologous FCXM (OR = 4.79,p = 0.006). Interestingly,patients on peritoneal dialysis (PD) also had significantly more positive autologous FCXM than patients on hemodialysis or waiting for pre-emptive kidney transplants (OR = 3.27,p = 0.02). These findings were confirmed in patients who had false positive allogeneic FCXM. Twenty of 24 (83.3{\%}) patients with false positive allogeneic FCXM tested with updated method either had immunological diseases originally or were on PD. Our findings are helpful when interpreting an unexpected positive FCXM,especially for transplantation from deceased donors. View Publication

Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis,binding and uptake of exosomes are well characterized4,5,delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology,we show that these two lipid receptors,which interact in the late endosome9,are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway,which Ebola virus exploits to infect cells9,mediates HAV infection by exo-HAV,which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen,but not the endosomal uncoating of HAV particles contained in the exosomes,is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV,infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses,and call for a reassessment of the role of envelope glycoproteins in infection. View Publication